Escherichia coli's main efflux pump AcrB, drug efflux occurs in the porter domain, while protons flow through the transmembrane domain: remote conformational coupling. At the border of a transmembrane helix (TM8) and subdomain PC2, there is a loop which makes a hoisting movement by a random-coil-to-α-helix change, and opens and closes a drug channel entrance. This loop is supposed to play a key role in the allosteric conformational coupling between the transmembrane and porter domain. Here we show the results of a series of flexibility loop-mutants of AcrB. We determined the crystal structure of a three amino acid truncated loop mutant, which is still a functional transporter, and show that the short α-helix between Cβ15 and the loop unwinds to a random coil in the access and binding monomers and in the extrusion monomer it makes a partially stretched coil-to-helix change. The loop has undergone compensatory conformational changes and still facilitates the opening and closing of the channel. In addition, more flexible mutated loops (proline mutated and significantly elongated) can still function during export. The flexibility in this region is however limited, as an even more truncated mutant (six amino acid deletion) becomes mostly inactive. We found that the hoisting-loop is a highly flexible hinge that enables the conformational energy transmission passively.The overexpression of RND-type exporters is one of the main causes of multidrug resistance (MDR) in Gram-negative pathogens. In RND transporters, such as  Escherichia coli, the main RND transporter is AcrB, part of the AcrAB-TolC complex  is a serious problem in global health we face today  and six C-terminal TM-helices (TM7-TM12) are twisted relatively to each other as a result of the ion-pair breakage between Asp407, Asp408 and Lys940. During this transition to the extrusion monomer, residue Arg971 extends and bends downwards to TM10 and the phenyl-ring of Phe948 is tilted downwards and Met970 bent away. In this way, Arg971 is exposed to a water-void open to the cytoplasm and can release a proton. Now, in the extrusion monomer, the loop at the N-terminal end of TM8 performed a hoisting-like motion by its conformational change from a random coil to an α-helix (to make an extended TM8), presumably playing a key role in conformational transmission and facilitating the motion of PN1/PC2 during the transition to the extrusion monomer . In the extrusion monomer, this segment forms three turns of the α-helix which extends the top of TM8. On the other hand, in the access and binding monomer, a part of this helix (868LSGNQ872) unwinds to a random coil, enabling the upward swinging motion of PC2 by extending the distance from TM8 to Cβ5. This random-coil-to-α-helix change also opens and closes the membrane surface channel (CH1) during the drug extrusion cycle  of the PC2 subdomain and TM8   .The minimal inhibitory concentrations (MIC) for wild-type and mutant AcrB were determined using LB-agar plates supplemented with substrates. The substrates were added in a series of dilutions. Agar plates were stamped with the desired strains and grown at 37°C overnight. The MIC values are the concentration of drugs in the agar plates at which no cells were viable anymore. For growth curves, cells were grown from diluted exponentially growing cells in 96-well plates for 8 h at 37°C. ODacrB E. coli cells were used. Overnight cultures of the E. coli cells were diluted and grown at 37°C until OD600nm 0.5–0.6 was reached. Cells were harvested and washed twice with Efflux Buffer (100 mM potassium phosphate (pH 7.5) and 5 mM MgSO4) and diluted to final OD600nm of 18. 10 μM of ethidium bromide and 10 mM arabinose was added. Ethidium bromide fluorescence was measured by SH-8100 reader (Corona Electric Co.) using λex = 530 nm and λem = 600 nm. Exclusion assays were repeated at least four times providing the error envelopes.For ethidium bromide exclusion efflux determination, BL21ΔacrB E. coli cells harboring the pAcBH plasmid. The final mutant AcrB buffer condition was 20 mM sodium phosphate pH6.2, 10% (v/v) glycerol, 0.05% (w/v) n-Dodecyl-β-D-maltoside and the final protein concentration was 20 mg/ml. Crystals of AcrB variants were made by the hanging drop vapor diffusion method at 25°C. The protein solution was mixed with an equal volume of reservoir solution. The reservoir solution contained 100 mM SPG  buffer at pH5.0 and 20% (w/v) PEG1500.C-terminally His6-tagged mutant AcrB (hoisting-loop truncated mutant) were expressed in JM109ΔiMosflm . X-ray data set was indexed, integrated and scaled using a Evans,  from CCP860TGMSYQERLSGNQ872 located at the N-terminal end of TM8 . We tested the efflux ability of the mutated pump by determining the growth ability of AcrB-expressing E. coli cells (both wild-type and mutant) in the presence of various structurally unrelated compounds: minocycline, erythromycin, ethidium bromide, cloxacillin, acriflavine, benzalkonium, crystal violet and rhodamine 6G. We also tested the truncated mutant on ethidium bromide exclusion ability in E. coli cells, compared to wild-type AcrB expressing and acrB-knockout cells. When ethidium enters the cell, it binds to DNA and becomes fluorescent. Active AcrB transporters export ethidium out of the cell from the periplasm or outer leaflet of the inner membrane before it enters the cytoplasm and the fluorescence is kept significantly low, hence acrB-knockout cells give a high fluorescent signal. The mutant was expressed equally to wild-type AcrB . In general, the structure is almost identical to wild-type AcrB . Surprisingly, the structure shows that the entrance of CH1 to the proximal binding pocket is still completely open in the access and binding monomers Figure , similar Figures . As seen Figures , althoug Figures . It also868LSGGSGNQ, 868LSGGSGGSGNQ, and 868LSGGSGGSGGSGNQ , as depicted in Figure Q Figure . In addip Figure . We testp Figure .We show that the hoisting-loop is a highly flexible hinge that enables the large conformational changes of the subdomains of AcrB. Helix-breaking proline mutants, elongated loop mutants and truncated mutants show that this loop is extremely flexible. These protein mutants are still able to execute the required drug extrusion cycle Table . The resCrystal structures of AcrB TM-mutants by Eicher et al.  support 1-ATPase, which also comprises similar functional access, binding and extrusion states by its monomers. Perhaps similar to F1-ATPase  exhibit the same phenotype as wild-type AcrB. As there are no other significant conformational changes in loops and regions linking the TM- and porter-domains directly during the drug extrusion cycle , another hypothesis needs to explain remote energy coupling mechanism of RND transporters. Computer simulations may shed a light on the complex mechanism of the exporter. Energy transduction might be due to the subtle changes in the TM-domain, explained by packing efficiency through the solvent-entropy effect translated to the porter domain, described by thermodynamics by computer simulations (Mishima et al., In summary, we show that the hoisting-loop is extremely flexible and necessary. Even a truncated loop mutant is able have a functional gating mechanism with an open CH1 channel, although additional truncation significantly inhibits the function of the exporter. Fully active elongated mutants show us that this region of AcrB is extremely flexible and that an increase of flexibility does not inhibit the energy transduction nor the function of the protein. The flexible loop can facilitate the significant changes in the porter subdomains PC1 and PC2. Our knowledge and understanding of RND transporters is crucial for the development of new antibiotic therapeutics.5YIL.MZ and KH performed the molecular biological and biochemical experiments. KK, MZ, KH, RN, and KS performed the protein crystallization and structure analysis. KH and MZ designed the research. MZ, KN, and AY wrote the manuscript. The coordinates for the hoisting-loop mutant of AcrB have been deposited in the Protein Data Bank under accession number The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Rattus norvegicus have not been studied. In the present study, we performed deep sequencing of small RNA molecules in the embryonic fibroblasts, embryonic stem cells, and induced pluripotent stem cells of laboratory rats. Bioinformatics analysis revealed 674 known miRNAs and 394 novel miRNA candidates in all of the samples. Expression of known pluripotency-associated miRNAs, such as the miR-290–295 and miR-183-96-182 clusters as well as members of the miR-200 family, was detected in rat pluripotent stem cells. Analysis of the targets of differentially expressed known and novel miRNAs showed their involvement in the regulation of pluripotency and the reprogramming process in rats. Bioinformatics and systems biology approaches identified potential pathways that are regulated by these miRNAs. This study contributes to our understanding of miRNAs in the regulation of pluripotency and cell reprogramming in the laboratory rat.MicroRNAs (miRNAs) constitute a class of small noncoding RNAs that plays an important role in the post-transcriptional regulation of gene expression. Much evidence has demonstrated that miRNAs are involved in regulating the human and mouse pluripotency. Nevertheless, to our knowledge, miRNAs in the pluripotent stem cells of one of the most commonly used model organisms – the  PSC fate is regulated by a complex network including transcription factors, signalling pathways, epigenetic modifications, and non-coding RNAs8. The regulatory pathways of self-renewal and pluripotency are not completely understood, especially in rats. The laboratory rat is widely used in various biomedical research fields such as disease modelling, pharmacology and toxicology9. However, the absence of rat PSCs has, for a long time, limited its further application in biomedicine. Rat pluripotent cells were first derived in 2008 using special serum-free conditions, leukaemia inhibitory factor, and inhibitors of the MEK1/2 and GSK3 kinases11. It is known that PSCs have species-specific properties and that microRNAs (miRNAs) play a substantial role in the regulation of pluripotency15.Pluripotent stem cells (PSCs) are a unique model for studying early development, disease modelling, and use in regenerative medicine. PSCs are capable of self-renewing and differentiating into all cell types of the adult organism. Two types of pluripotent cells exist – embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). ESCs are derived from the inner cell mass of blastocysts, and iPSCs are generated from somatic cells by the overexpression of Yamanaka factors 17. miRNAs are critical for the regulation of self-renewal and pluripotency, as was shown by knockout studies of Dicer and DGCR8 – enzymes that are involved in miRNAs processing21. Some miRNAs, such as miR-291, miR-294 and miR-295, can enhance reprogramming that is induced by OSK factors22. Moreover, overexpression of a miR-302-367 cluster was sufficient to reprogramme human and mouse fibroblasts without overexpressing the Yamanaka factors23.miRNAs are small 18–23-nt non-coding RNAs that regulate gene expression by mRNA degradation or translation inhibition24 as well as six different tissues, including spleen, liver, brain, testis, heart, kidneys25. Recently, miRNA analysis was performed on the 21 and 23 organs of male and female Sprague Dawley rats, respectively26. However, miRNA expression in rat PSCs has not been studied yet. We recently derived rat ESCs and iPSCs and characterized their transcriptional profile27. All of our derived ESC and iPSC lines were AP-positive, expressed key pluripotency markers, and were able to differentiate into derivatives of all three germ layers in vitro (embryoid bodies) and in vivo (teratoma test). Female iPSC lines are characterized by two active X-chromosomes. The rat ESCs and iPSCs were also able to contribute to the tissue development of chimeric organisms in the xenogenic rat-mouse model. In this study, we performed next generation sequencing of small RNA libraries from rat ESCs, iPSCs and embryonic fibroblasts (EFs). We analysed miRNA expression in these cell types and identified novel rat miRNAs that were expressed in PSCs and EFs. Using the KEGG signalling pathways and Gene Ontology (GO) enrichment analyses of known and novel miRNA targets, we revealed the involvement of miRNAs in the regulation of pluripotency and cell reprogramming. These findings provide new insights into pluripotency network regulation in rats.Previous genome-wide sequencing studies of rat miRNAs have been conducted using adult tissues. Rat miRNAs were sequenced and analysed from several neural compartments and cell lines, such as thyroid FRTL-5 cells and their differentiated derivatives27. EFs were isolated from E12 rat embryos (WAG and Brattleboro strains). ESCs were derived from the inner cell mass of E4.5 blastocysts as described previously11. iPSCs were produced from rat EFs by the overexpression of Oct4, Sox2, Klf4 and Myc using a lentiviral doxycycline-inducible system28. Cells were cultivated as described27. EFs were maintained in Dulbecco’s modified Eagle’s medium (DMEM) mixed with Ham’s F12 medium (1:1), containing 10% foetal bovine serum, GlutaMAXTM, 5,000 U/mL penicillin/streptomycin, nonessential amino acids, sodium pyruvate, and 0.1 mM 2-mercaptoethanol. ESCs and iPSCs were cultivated in N2B27 medium containing GlutaMAXTM, 5,000 U/mL penicillin/streptomycin, 0.1 mM 2-mercaptoethanol, 1,000 U/mL mouse LIF (StemRD), 1 µM PD0325901 (StemRD), and 3 µM CHIR99021 (StemRD). ESC and iPSC differentiation was performed for 24 days on tissue culture surfaces coated with gelatine according to a previously published protocol27. Cells were disaggregated with collagenase IV or TrypLE and were seeded on a gelatine-treated surface at a density of 104 cells/cm2 in medium containing a 1:1 mixture of N2B27  and DMEM/F12 media supplemented with 10% foetal bovine serum, nonessential amino acids, GlutaMAX, 10 ng/mL basic fibroblast growth factor, and 10 mM Y27632 (R&D).Rat EFs, ESCs and iPSCs were previously obtained and characterized in our labCells were resuspended in RNAlater and used for small RNA extraction with the mirVana™ miRNA Isolation Kit . RNA quality was analysed using an Agilent Bioanalyzer. A 50-ng quantity of small RNA was used to prepare a library, which was then sequenced on the Illumina GAIIX. Raw data were deposited into the SRA database with the accession numbers SRX395433-395442.Rnu1a expression.Small RNA for cDNA synthesis were extracted from cells and tissues with the mirVana™ miRNA Isolation Kit. cDNA was synthesized by the NCode™ VILO™ miRNA cDNA synthesis Kit (Invitrogen) from 1 µg of total RNA. Real-time PCR was performed using the HS-qPCR SYBR Blue mix (Biolabmix) with a custom miRNA-specific primer and a universal primer from Invitrogen on the LightCycler480 System (Roche). Reaction conditions were 95 °C for 5 min followed by 40 cycles of 95 °C for 15 secs and 60 °C for 30 sec. Reaction specificity was verified by melting curve analysis. Reactions were performed in triplicate. Sequences of the miRNA-specific primers are provided in Supplementary Table Raw reads of 11 samples were processed with the FASTX Toolkit v0.0.14 in two steps. First, the data were trimmed with the fastq_quality_trimmer tool, leaving only reads with a tail quality of at least 30. Then, an adapter sequence was clipped with the fastx_clipper program, leaving only 15-nt reads or longer.R. norvegicus reference genome (version rn5) from the UCSC Genome Browser32 combined with the ribosomal RNA reference for R. norvegicus from the Silva database33. The BWA v0.7.10-r806 software was utilized for gapped/ungapped alignment of single-ended reads. If a read could be mapped to several places in the reference, only one random place was chosen. The portion of reads that mapped solely to chr1-20,X varied from 94.2% to 97.4% in different samples.Filtered reads were mapped to the R. norvegicus was downloaded from miRBase v21 (ftp://mirbase.org/pub/mirbase/CURRENT/genomes/rno.gff3)34. The absolute majority (99%) of reads that mapped to the genomic intervals of pre-miRNA had a length of 18–27 nt. To keep the focus of this study on miRNA, we filtered out reads that were shorter than 18 and longer than 27 nt because they likely represented short RNA species, which are different from the miRNA class. The rest of the reads were further filtered from the ones mapped onto parts of the genome that is annotated with rmsk repeats (http://hgdownload.soe.ucsc.edu/goldenPath/rn5/database/rmsk.txt.gz)29 of class “RNA” . This filtration did not influence any reads that were mapped to known miRNAs. A final set of mapped and filtered reads was called informative reads. A summary is presented in Table To assess the quality of reads, a set of 486 genomic intervals of known premature miRNAs for informative reads for two separate groups of samples: PSCs and EFs. The wig files contained RPM (reads per million) expression values for each position on each chromosome. RPM is calculated as the number of reads overlapping a position in the genome divided by the total number of informative reads in a group of samples. Then, wig files were inspected for genome intervals with specific double-peak signals, as explained in Supplementary Figure 29, and (3) RNAFold program35 predicted a hairpin structure. As a result, 73 potential pre-miRNAs remained. Following further analyses of sequence homology, some of them were found to belong to the snoRNA class and had to be analysed separately.Wig files were created from the genome maps of the 36 was applied to each sample and entire dataset of 11 samples. The R. norvegicus genome (assembly version rn4) was used. The default parameters were used except for the “Fast detection of novel microRNAs”, which was unmarked. For each sample and separately for entire dataset of 11 samples, potential miRNAs that were named “perfect Dicer” were selected from the predictions. Then, only “perfect Dicers” that did not intersect with genome elements (empty “genome elements” column) were selected. Potential miRNA coordinates in rn4 were converted to rn5 using the UCSC LiftOver tool and then intersected with refSeq genes (“refGene.txt” downloaded from UCSC on 14.04.15). Only the entries that did not overlap the refSeq genes in rn4 and rn5 were kept. If a potential miRNA was found in at least two samples, it was labelled as a “cluster” miRNA. The longest or the most strongly expressed isoform was selected as a representative of the cluster. Potential novel miRNAs that were found by miRanalyzer and were predicted35 to have a hairpin structure were filtered against known rat miRNAs (ftp://mirbase.org/pub/mirbase/CURRENT/genomes/rno.gff3)34 and RNA repeats (http://hgdownload.soe.ucsc.edu/goldenPath/rn5/database/rmsk.txt.gz)29. As a result, 13 potential pre-miRNAs remained.The miRanalyzer program37 was launched on the collapsed genome (rn5) maps of informative reads. A training set of known mature miRNAs included R. norvegicus (ftp://mirbase.org/pub/mirbase/CURRENT/genomes/rno.gff3)34 and Mus musculus (ftp://mirbase.org/pub/mirbase/CURRENT/genomes/mmu.gff3)34 miRNA sequences acquired from miRBase v21. Additionally, a set of miRNA hairpin sequences was taken from miRBase v21 (ftp://mirbase.org/pub/mirbase/CURRENT/hairpin.fa.gz)34. Potential novel miRNAs found by mirDeep2 were filtered against known rat miRNAs (ftp://mirbase.org/pub/mirbase/CURRENT/genomes/rno.gff3)34 and overlapping RNA repeats (http://hgdownload.soe.ucsc.edu/goldenPath/rn5/database/rmsk.txt.gz)29, which resulted in a list of 315 potential pre-miRNAs.MiRDeep2 v2.0.0.729, with consideration of the DNA strand.We took advantage of the UCSC genome browser tracks of rat and other species’ genes, including miRNA, that were mapped to the rn6 genome. To find homologs of novel miRNAs, we intersected their genomic intervals with exons and introns of the ensGene track  and the xenoRefGene track  (http://hgdownload.soe.ucsc.edu/goldenPath/rn6/database/)34. The number of All mapped reads overlapping each miRNA genome interval, with consideration of DNA strand, was calculated in each sample. If a read overlapped the border of an interval, it was counted as 1. The sum of overlapping reads served as a raw digital expression level for each miRNA. Alternatively, raw expression was calculated for the informative reads (a subset of All reads that were 18–27 nt in length without RNA-type repeats). The raw expression of known miRNAs calculated by an alternative method was only 1% less than the expression that was calculated based on All mapped reads. To be less stringent, we took the expression level that was based on All reads for downstream analyses. In order to account for the different initial sequencing depths of samples, the RPM normalization procedure was applied to the raw expression values:i – number of informative reads in sample i informative reads to the number of All mapped reads was used to assess the portion of 18–27 nt reads. If this ratio was less than 0.5, the novel RNA molecule was moved to Tier 2. If the ratio was between 0.5 and 0.85 and the difference between the counts of All and informative reads was higher than 3, the pre-miRNA was also moved to Tier 2. The second classification parameter was calculated for novel pre-miRNA candidates in which the genomic interval was 50 nt or longer. Such intervals were divided into left and right parts of equal length. The parameter was used to assess the level of expression of the stem loop. It was calculated as the ratio m/(l + r), where l and r are the number of reads overlapping the left and right parts of the interval, respectively , and m is the number of reads overlapping the border between the left and right parts. If the ratio was higher than 0.05, the potential pre-miRNA was moved to Tier 2.By default, all potential pre-miRNAs were located in Tier 1. For each item, the following quality scores were calculated in order to sort them into two different tiers. First, the ratio of the number of http://www.R-project.org/)39, data were centred around the mean expression values, scaled to unit-variance and then processed using PCA40.RPM expression data of 832 known miRNAs, excluding 158 zero-expression entries, were used. Additionally, 304 novel miRNAs with unique sequences and not homologous to known miRNAs were used independently. Using the R statistical computing program  expression counts. Parameters of overdispersion were fitted by the software, taking into account the common dispersion, trended dispersion and tagwise dispersion of the miRNA expression values. A set of 227 miRNAs satisfied a p-value cut-off of 10−4, which corresponded to the 3.0 × 10−4 false discovery rate (Benjamini-Hochberg). Alternatively, ANOVA was also performed using the ‘aov’ function in R after replacing zero values with uniform random values from interval (0 to 0.1) and log2 transformation  higher than its maximum level in PSCs, and additionally, they had to have at least three mapped reads in each of the EF samples. Only 219 miRNAs passed this procedure of selection, and they had an edgeR p-value of less than 0.01. Differentially expressed novel miRNAs were defined in the same way.ESC and iPSC groups were combined together (PSCs) and compared with EFs. P-values were calculated with the edgeR softwareExpression values of zero were replaced with 0.25, which is below the minimum positive value. After that, log2 transformation was applied. The clustering of miRNAs and samples based on expression values was performed using the ‘pheatmap’ function in R using the default parameters.42 . Lists of the targets of differentially expressed known miRNAs were obtained taking the miRNA names as the key. They were additionally filtered using gene expression data obtained from sequencing experiments that were performed on the same biological samples27. Upregulated targets were kept if the corresponding miRNA was downregulated, and downregulated targets were kept if the corresponding miRNA was upregulated . After filtration against RNA-type repeats and after keeping only 18–27 nt sequences, a set of 8,860,854 informative reads was obtained. The exact amount of informative reads for each library is presented in Table informative reads was similar among different samples. Examples are given in Fig. informative reads were between 21–25 nt. To identify known miRNAs, coordinates of All reads that were mapped to the genome were compared with known miRNA coordinates from miRBase v2134. The portion of known miRNA reads , two ESC , and seven iPSC samples  were generated and sequenced on the Illumina platform. Reads were filtered as described in the Materials and Methods section. A total of 32.9 million raw reads were generated by the sequencing of eleven libraries followed by trimming and adapter clipping. An absolute majority (31.9 million raw reads) mapped onto the reference genome, including 31.4 million reads that mapped on the chr1-20, X . Homology analysis was done by overlapping genome intervals with the xenoRefGene and ensGene UCSC genome tracks29. This analysis allows nucleotide gaps, deletions and mismatches between homologs, including variance in miRNA seed sequence. The rest of the 309 candidates were split into Tier 1 and Tier 2 lists according to their quality scores as described in the Materials and Methods section and each had 248 and 61 items, respectively. Tier 1 and novel species that are homologous to known miRNAs are presented in Supplementary Table To discover novel miRNA species, the All mapped reads as described in the Materials and Methods section. Among the known miRNAs, 77 were found to be upregulated and 142 were found to be downregulated in PSCs compared to EFs, with at least a double extremum fold change  gene expression was calculated for the 832 known and the 557 potential novel miRNAs using extremum fold change of at least 10) and on all expressed miRNAs. Figure PCA was performed based on the RPM values of 219 differentially expressed known miRNAs. Figure 47. Several miRNAs, such as members of the miR-183-96-182 cluster, miR-741, miR-881, miR-878, miR-743a, miR-743b, miR-465, miR-871, miR-880, are known to be expressed in mouse ESCs and were also expressed in rat PSCs; however, their function in pluripotency induction and regulation has not been directly shown50.A number of miRNAs that were found to be upregulated in rat PSCs, such as the miR-290-295 cluster and the miR-200 family miRNAs, are involved in pluripotency induction and maintenance in miceextremum fold change of more than 10 between EFs and PSCs. Among them, 58 miRNAs were upregulated in the PSCs and 69 were upregulated in the EFs ; chr7:+:70463555–70463594 (rno-homolog-miR-26); chr3:+:16697111–16697143 (rno-homolog-miR-199); and chr18:−:69422790–69422857 (rno-miR-sno-57) were selected for the validation. rno-miR-novel-8, rno-homolog-miR-26, and rno-homolog-miR-199 miRNAs were selected from Tier 1, and rno-miR-sno-57 miRNA was selected from Tier 2. In addition, we analysed the expression of miR-741-3p and miR-743a-3p and found that, in accordance with sequencing data, they were highly expressed in rat PSCs. A pluripotency-associated miRNA, miR-295-3p, was used as a positive control. Expression of miR-295-3p was observed in rat PSCs and absent in rat EFs, which was consistent with its expression pattern in the mouse51 Fig. . qRT-PCR51 Fig. . rno-hom51 Fig. . For nov51 Fig. . We also51 Fig. .Figure 4R. norvegicus is one of the most promising models for disease modelling and translational biomedicine. Pluripotent stem cells are currently widely used to generate various disease models52. An understanding of pluripotency and self-renewal regulation is required to improve the approaches to PSCs generation and cultivation, which will help realize the full potential of their application. Although much progress has been made in the understanding of the pluripotency regulatory network, it has not been fully investigated yet, especially in rats. miRNAs are known to play substantial roles in the regulation of pluripotency and self-renewal14. To study miRNA expression in rat PSCs and to find novel rat miRNAs, we performed genome-wide sequencing of small RNA libraries from rat ESCs, iPSCs, and EFs. Analysis of the sequencing data revealed the expression of 674 known miRNAs across all samples. Differential expression analysis of known miRNAs demonstrated that 77 miRNAs were upregulated and 142 miRNAs were downregulated in rat PSCs compared to EFs. Members of the miR-290-295 cluster  were the most abundant among known miRNAs that were upregulated in rat PSCs. It is known that mouse miR-290-295 and its human homologues in the miR-372 clusters are ESC specific and regulate cell cycle progression21. Among the miRNAs upregulated in rat PSCs, we also identified miR-205 and members of the miR-200 family , which promote mesenchymal to epithelial transition (MET) in mouse cells, a key step in fibroblast reprogramming47. Members of the miR-200 family are known to suppress the Zeb2 transcription factor, and its inhibition promotes MET53. Another miRNA cluster, miR-183-96-182, is known to be expressed in mouse PSCs49 and is also expressed in rat PSCs. The microRNA-183-96-182 cluster inhibits epithelial to mesenchymal transition (EMT) in cancer cells, and thus, the miRNA cluster may facilitate the reprogramming process by promoting MET57. Members of the miR-106-363 and miR-106b-25 clusters, which were shown to be upregulated during fibroblast reprogramming in mice58, were also upregulated in rat PSCs. Most of the miRNAs upregulated in rat PSCs are also expressed in mouse ESCs or iPSCs48. In addition, we found several rat miRNAs  whose expression had not been established in PSCs before.59. These miRNAs are testis specific in rats, according to the miRNA sequencing data of 21 male rat organs26. Using qPCR, we have shown that members of the miR-741-3p and miR-743a-3p clusters were expressed at high levels in rat ESCs and iPSCs and were downregulated during differentiation. These miRNAs are also expressed in some adult rat tissues, according to our data and RT-PCR expression profiling with the TaqMan® Rodent miRNA card A v2.0 .A cluster of miRNAs, comprised of miR-463, miR-465, miR-471, miR-741, miR-743a, miR-743b, miR-871, miR-878, miR-880, miR-881, and miR-883, was located on the X chromosome and was expressed at a high level in rat PSCs according to the sequencing data. These miRNAs are known to be expressed in mouse ESCs and testes60. However, expression levels of miR-302a and miR-302b were low in rat PSCs compared to the ESC specific miR-290-295 cluster. This is in agreement with the fact that high expression levels of miR-302 cluster are typical of mouse epiblast stem cells48. We also found a homologue of human mir-372 located inside the rat miR-290-295 cluster. miR-372 in humans is known to be connected to fibroblasts reprogramming, particularly by promoting MET60.The number of known rat miRNAs is much lower compared to that in humans and mice. In this study, 394 novel mature miRNAs were identified using three methods – mirDeep2, miRanalyzer, and custom peaks2mirna. Most of the novel miRNAs were identified by the mirDeep2 method. In rat PSCs and EFs, 23 novel miRNAs were differentially expressed. Among them, 10 novel miRNAs were upregulated and 13 were downregulated in PSCs. This list includes homologues of human miR-302a and miR-302b. We found that these miRNAs were upregulated in rat PSCs. We also found a homologue of human miR-367. The human and mouse miR-302-367 cluster has been demonstrated to play an important role in the regulation of pluripotency. Moreover, overexpression of the miRNA cluster was sufficient to reprogramme fibroblasts to iPSCs51. In addition, we found that the rno-miR-novel-8 miRNA was located less than 10 kb from miR-871, mir-3580 and miR-465, which were upregulated in rat PSCs. This may be a cluster of miRNAs that is involved in the regulation of pluripotency, and further investigations are required to shed light on its function.Four novel miRNAs were validated using qRT-PCR analysis. Three miRNAs were selected from Tier 1, and one was selected from Tier 2. Expression of the two validated novel miRNAs was observed in rat PSCs and was absent in EFs. Expression of rno-miR-novel-8 miRNA decreased during differentiation and was not observed in the analysed adult organs, except in the testes. The pattern is similar to ESC-specific miRNAs, such as members of the miR-290-295 cluster61. To better understand the functions of known rat miRNAs that are differentially expressed between PSCs and EFs, we analysed their target genes that have been validated in rats, humans or mice. An additional criterion was that the change in the PSC and EF target gene expression levels had to be opposite of that for miRNAs. This criterion helped to improve the analysis of miRNA functions in the rat. In total, 388 downregulated targets were found for the upregulated miRNAs in PSCs, and 69 upregulated targets were observed for miRNAs that were downregulated in PSCs. Then, we performed KEGG and GO enrichment analyses of the target genes. The most common GO terms were related to transcriptional regulation, which indicated that a substantial part of the target genes functioned as transcriptional factors. Many GO terms, such as stem cell differentiation and response to retinoic acid, were related to cell differentiation, which was consistent with the function of miRNAs in suppressing PSCs differentiation62. Self-renewal and differentiation processes are balanced in PSCs, and miRNAs are involved in the regulation of this process61. We also found a stem cell population maintenance category that was relevant for targets of miRNAs that were differentially expressed in PSCs. Regulation of the apoptotic process was also an important category for pluripotent cells, because inhibition of apoptosis promotes pluripotency63 and was demonstrated for the ESC-specific miR-290-295 cluster64. We found enrichment of differentially expressed miRNAs and their targets in the KEGG signalling pathway, the stem cell pluripotency regulatory pathway, pathways in cancer, and the Hippo signalling pathway. Pathways in cancer are relevant for miRNAs that are upregulated in PSCs. It is known that miRNAs that are expressed in PSCs are frequently related to different types of cancer. The human miR-372-373 cluster promotes tumorigenesis, for example65. The Hippo signalling pathway represents a barrier for reprogramming, and its inhibition by LATS2 knockdown increases the efficiency of iPSC generation66. Analysis of the putative target genes of novel rat miRNAs has shown that the miRNAs may take part in the regulation of development and differentiation. In addition, KEGG signalling pathway analysis suggests the involvement of miRNAs in the regulation of the Wnt signalling pathway, which regulates rat PSC maintenance, and the TGF-beta pathway, which participates in the regulation of cell reprogramming47. Based on the results, we suggest that the rat miRNAs identified in this study are involved in the regulation of pluripotency and reprogramming in rats.miRNAs implement their functions through repressing target gene expression. In human and mouse PSCs, clusters of pluripotency-associated miRNAs control cell proliferation and prevent differentiationIn conclusion, we first compared the miRNA expression in rat embryonic stem cells and induced pluripotent stem cells with rat embryonic fibroblasts used for reprogramming. The miRNA expression signature in rat PSCs was similar to that in mouse PSCs, and most mouse pluripotency-associated miRNAs were also found in rats. A number of novel rat miRNAs were identified. Overall, our results shed light on the role of miRNAs in the pluripotency regulatory network of the laboratory rat.Supplementary InformationSupplementary Table S1Supplementary Table S2Supplementary Table S3Supplementary Table S4Supplementary Table S5Supplementary Table S6Supplementary Table S7Supplementary Table S8Supplementary Table S9


To the Editor:in vivo kinetic response. We therefore investigated the kinetics of allergen-induced activation of the complement and coagulation cascades in nasal mucosal lining fluid.The interlinked pathways of complement activation, coagulation, fibrinolysis, and fibrosis contribute to numerous respiratory diseases.www.jacionline.org. Two phases of the allergic response have been described: the early allergic reaction  and the late allergic reaction .A), probably resulting from mast cell activation; these kinetics closely match a previous observation of β-tryptase release by mast cells.A). All data in www.jacionline.org. The 30-45 minute peak of complement activation was universal among volunteers, where such early peaks likely represent activation of complement preexistent in the airway. In contrast, the second peak was clearly evident only in some volunteers. Matrix metalloprotease 9 (MMP9) and the type II mediators IL-5 and IL-9 were significantly elevated in the LAR, peaking at 480 minutes (B).The NAC protocol, demographic characteristics, and clinical response to challenge are summarized in the Results section and  minutes , B.Fig 1A, in this article's Online Repository at www.jacionline.org). These data suggested that coagulation can occur in the upper airway without the need for local TF production post-NAC. Levels of intermediates of coagulation were below detection limits  using a TF-dependent thrombin generation assay, but levels were unaltered by NAC (see  see the  section.C). Similarly to complement, late-phase D-dimer elevation was evident in only approximately 50% of participants. Fibrinolysis is plasmin-dependent and the plasminogen activation cascade has been associated with asthma and allergies,C; see I, in this article's Online Repository at www.jacionline.org).Coagulation generates fibrin that is cleared by fibrinolysis, resulting in the formation of fibrin degradation products including D-dimer. Strikingly, D-dimer levels also increased in a biphasic manner, reminiscent of complement components, with peaks observed at 45 and 480 minutes after NAC , C. SimiA). The P values and Spearman R scores of this matrix are listed in www.jacionline.org.A correlation matrix was established to determine the interrelation between markers of mast cell activation, complement activation, type II inflammation, and fibrinolysis during the EAR and LAR. The matrix analyzed area under the curve values of the induction of mediators over the EAR and LAR , A. The P = .04; A). Trends were observed between C5a and D-dimer levels, in both the EAR and LAR . LAR levels of C5a correlated with u-PA and IL-5 . In addition, a strong correlation was observed between IL-5 and D-dimer , where proteases such as MMP9 could activate complement. In asthma, sputum eosinophils have been reported to correlate with D-dimer levelsThis study details the kinetics of complement activation and fibrinolysis in the upper airway following NAC, extending a previous report of gene expression.


Terpyridine and quaterpyridine-based complexes allow wide light harvesting of the solar spectrum. Terpyridines, with respect to bipyridines, allow for achieving metal-complexes with lower band gaps in the metal-to-ligand transition (MLCT), thus providing a better absorption at lower energy wavelengths resulting in an enhancement of the solar light-harvesting ability. Despite the wider absorption of the first tricarboxylate terpyridyl ligand-based complex, Black Dye (BD), dye-sensitized solar cell (DSC) performances are lower if compared with N719 or other optimized bipyridine-based complexes. To further improve BD performances several modifications have been carried out in recent years affecting each component of the complexes: terpyridines have been replaced by quaterpyridines; other metals were used instead of ruthenium, and thiocyanates have been replaced by different pinchers in order to achieve cyclometalated or heteroleptic complexes. The review provides a summary on design strategies, main synthetic routes, optical and photovoltaic properties of terpyridine and quaterpyridine ligands applied to photovoltaic, and focuses on n-type DSCs. Dye-sensitized solar cells (DSCs) are photoelectrochemical devices able to convert sunlight into electricity . The arcSince 1997  the inteet al. . Th. Thet alties 18,  68] but butet al2g orbital  [I] / 1-ethyl-3-methylimidazolium tetracyanoborate 65:35). Similar bipyridines, slightly modified in the styryl substitution, were also tested by Giribabu et al. ..45, 146]et al. [46, 3CN / n-butanol 1:1 with 20 mM DCA, electrolyte: 0.5 M DMPII, 0.05 M I2, 0.1 M LiI CH3CN / butanol 1:1).A bidentate benzimidazole was tested by Swetha et al.  as ancil3MLCT states. No significant differences in IPCE values were found in the case of the various Os complexes showing values up to 50% at 900 nm and 70% in the visible region. A better stability was ascribed to Os complexes with respect to the Ru case, even though these complexes showed lower light conversion.Terpyridine complexes with other metals were reported by Bignozzi’s group, who complexed osmium with tctpy, various bipyridines and pyridylquinoline ,149,150.2. An improved stability of the ruthenium dye was reported, even if these structures have not been tested on DSCs devices. More recently, Duchanois [2 photoanodes (homologous to the Ru complex 43a), and compared it with a bis-tpy iron complex  complexes usually display an intense charge-transfer absorption band in the visible region, Kwok et al. , in 2010et al. [scJ was detected, nevertheless a 2.16% efficiency and long lived excited-state lifetime were reported.Shinpuku et al.  synthesi10 metal ions complexes such as Zn-porphyrines has grown in photonic applications, ranging from OLEDs and LECs to DSCs technologies. Bozic-Weber et al. [2 sensitization. Terpyridines substituted with various anchoring and triphenylamino moieties extended with benzothiadiazole-diphenylamino units gave efficiencies between 0.5% and 1%. Housecroft et al. reviewed sensitizers made of Earth-abundant metals, concerning copper [The interest for dr et al. ,156,157 g copper  and otheg copper .Tpy complexes have been investigated also for the sensitization of p-type semiconductors. In p-type DSCs, the rules for sensitizers design are inverted with respect to classical n-type DSC cells. In fact, in these devices, the excited dye has to inject holes from HOMO to the conduction band of a p-semiconductor .et al., in 2013 [48, et al. [2 sensitization in solid state devices.Ji  in 2013 , propose, et al.  as bridg49, In 2014 both Constable  and Woodet al. [50, 2 mixtures with increasing amounts of polystyrene; 0.19 mM D131 and 0.56 mM BD in CH3CN / t-butanol 1:1, electrolyte: 0.15 M NaI, 0.075 M I2, 1.4 M DMPII, CH3CN / methoxyacetonitrile 9:1). Ozawa et al. [2 film with 45 μm thickness .The Black Dye has also been used in cocktail with other sensitizers characterized by higher molar extinction coefficient in the high energy regions of the spectrum, in order to increase the IPCE at lower wavelengths. Ogura et al.  used BD a et al.  optimizeSharma  proposedThe literature offers multiple choices in order to tune the photoelectrochemical properties of terpyridine-based complexes such as the Black Dye, ranging from the modification of the donor and acceptor ligands to the exchange of the metal center with other cations. The increase of the molar extinction coefficient has been commonly pursued by extending the π-conjugation on the ligands. Different anchoring moieties were compared, among which COOH turned out as one of the most effective groups. Isothiocyanate was often substituted by different ancillary ligands in order to improve long-term stability and the synthetic yield of complexation; bidentate and tridentate ligands that exploit coordination through N or C atoms have been tested in order to achieve a better sensitization. Tetradentate ligands have been used in order to further enlarge the spectral absorption properties.Few outlines can be depicted in this scenario for the design of future complexes: (1) better stability can be achieved avoiding the use of monodentate SCN ancillary ligands; (2) better performances are offered in the case of heteroleptic complexes (the homoleptic ones have an unfavourable symmetric charge distribution); (3) hydrophobic substitutions on the ligands are able to reduce the electron recombination; (4) a better coupling between the complex and semiconductor can be achieved when COOH moieties are used as attaching groups. Overall, a wise approach is requested in order to tune the energy levels far enough to reach panchromatic absorption, but not too much in order not to exceed the limit for a good regeneration rate by the electrolyte and a good electron injection driving force. Furthermore, the use of tpy complexes nowadays goes beyond the traditional role as sensitizers. Cobalt complexes have been reported as redox mediators, by exploiting the interaction of the EDOT-substituted complex with a PEDOT-covered counter-electrode  . By fine


The objective of the work was to develop a multifunctional nanomedicine based on a folate-conjugated lipid nanoparticles loaded with paclitaxel and curcumin. The novel system combines therapeutic advantageous of efficient targeted delivery via folate and timed-release of curcumin and paclitaxel via 2-hydroxypropyl-ß-cyclodextrin, thereby overcoming multidrug resistance in breast cancer cells (MCF-7/ADR). The faster release of curcumin from the folate-conjugated curcumin and paclitaxel-loaded lipid nanoparticles enables sufficient p-glycoprotein inhibition, which allows increased cellular uptake and cytotoxicity of paclitaxel. In western blot assay, curcumin can efficiently inhibit the expression of p-glycoprotein, conformed the enhancement of cytotoxicity by paclitaxel. Furthermore, folate-conjugated curcumin and paclitaxel-loaded lipid nanoparticles exhibited increased uptake of paclitaxel and curcumin into MCF-7/ADR cells through the folate receptor-mediated internalization. Taken together, these results indicate that folate-conjugated curcumin and paclitaxel-loaded lipid nanoparticles enables the enhanced, folate-targeted delivery of multiple anticancer drugs by inhibiting the multi-drug resistance efficiently, which may also serve as a useful nano-system for co-delivery of other anticancer drugs. Breast cancer is one of the most common cancers for women and still a major reason of death. Among breast cancer cell lines, MCF-7/ADR cells are known as multi-drug resistance (MDR) expressing cells. The development of MDR is one of the major challenges leading to the failure of several conventional chemotherapies , 2. The Curcuma longa Linn (turmeric). It has a variety of pharmacologic effects including anti-amyloid, anti-oxidant, anti-inflammatory, anti-bacterial, and anti-spasmodic activity without any major side effects [Paclitaxel, the microtubule-stabilizing agents, has been used as an effective chemotherapeutic agent for various solid tumors such as ovarian, breast, and lung cancers . Althoug effects –12. Spec effects , 14. Fur effects , 16. The effects .On the other hand, conventional co-delivery systems showed a few disadvantages such as unsuitable release profiles, use of conventional p-gp inhibitors which have toxicity to normal cells and non-targeted property. Hence, ideal delivery systems for combination should have targeting property and optimal release profile. Namely, the chance of exposure of anticancer agent and p-gp inhibitor to normal cells or tissues should be minimized and localized in a specific targeted cells or tissues. Furthermore, this system should use non-toxic p-gp inhibitor such as curcumin instead of conventional p-gp inhibitor such as verapamil and dronedarone that have toxicity . HoweverHerein, we have encapsulated curcumin in 2-hydroxypropyl-β-cyclodextrin (HPCD) to improve its stability and water-solubility for faster release relative to the release of paclitaxel Figure . As a reThe nanoparticles were fabricated by emulsification and the solvent evaporation method. The physicochemical properties including particle size, polydispersity index (PDI), zeta potential and encapsulation efficiency (EE) were listed in Table EE (%) of paclitaxel and curcumin in all of NPs observed to be above 75% except FPCHN-50. Interestingly, co-encapsulation of curcumin with paclitaxel in PCN resulted in slightly low EE of paclitaxel (81.3 ± 4.8%) as compared to that of PN (87.5 ± 3.5%). However, FA-conjugation did not impair EE of paclitaxel and curcumin. It was noted that the amount of HPCD used resulted in decreasing EE of curcumin, while EE of paclitaxel was not significantly changed. Specially, the EE of curcumin in FPCHN-50 was significantly lower than that of FPCN, while that of FPCHN-10 and -30 was insignificant different compare to that of FPCN.Surface modification of PCN with folate was confirmed using XPS. XPS result of PCN did not show nitrogen on the surface of NPs Table , while FIn vitro drug release of paclitaxel and curcumin from FPCN and FPCHN was conducted in PBS with 0.1% (w/v) tween 80. In order to achieve optimal sequential release profile of curcumin and paclitaxel, different amount of HPCD  was introduced to form inclusion complex with curcumin. As shown in Figure The quantitative cellular uptake of paclitaxel from the different formulations was presented in Figure In order to further investigate the uptake of curcumin in MCF-7/ADR cells, the fluorescence of curcumin was measured by confocal microscopy. This evaluation has been reported as the indirect method of nanocarrier penetration into the cells . As show3) and sucrose, were co-treated with FPCHN-30 in MCF-7/ADR cells, respectively. The introduction of NaN3 and sucrose reduced the mean intensity of curcumin to 38.7 and 26.5%, respectively.Flow cytometry analysis was carried out to figure out the cellular uptake mechanism of FPCHN-30 on MCF-7/ADR cells. Firstly, an effect of temperature on cellular uptake was tested by incubating the cells with FPCHN-30 at 4 or 37°C for 4 h Figure . The uptThe cytotoxicity of the different formulations against MCF-7/ADR cells was determined by the MTT assay for 24 and 48 h. As seen in Figure p < 0.01).Subsequently, the effect of folate targeting on the cytotoxic potential of the PCN, FPCN and FPCHN-30 on the MCF-7/ADR cells was evaluated Figure . As expeWestern blot assays were conducted to confirm the inhibition of p-gp expression in MCF-7/ADR cells treated with different formulations. As shown in Figure Immunofluorescence staining was further conducted to visualize the expression of p-gp Figure . In ordeMDR is a major limitation for a treatment of cancer because most of cancer-induced deaths are attributed to metastases that are resistant to anticancer drugs , 27. A fIn this study, a novel FA-conjugated targeting and sequential releasing of multiple drugs (i.e. paclitaxel and curcumin) loaded lipid nanoparticles (FPCHN) was developed for the smart approach on the efficient treatment in MDR expressing MCF-7/ADR cells. Paclitaxel is known as the first-line for the treatment of breast cancer. Recently, curcumin has been known as p-gp inhibitor as well as anticancer drug. Hence, it was hypothesized that first release of curcumin could inhibit p-gp and increase the cellular uptake of paclitaxel. A few studies have reported to overcome p-gp and enhance cytotoxicity by co-administration of a p-gp inhibitor with anticancer drug , 30. BesThe particle size range of all NPs was shown to be less than 200 nm except FPCHN-50. Such physical characteristics of all NPs in the range of 10-200 nm are likely advantageous to the NPs in exploiting the enhanced permeation and retention (EPR) effect against solid tumors through the leaky vasculature , 33. TheThe hydrophobicity can enhance EE and decrease release rate of drug in lipid nanoparticles. Hydrophobic drug can strongly interact with hydrophobic lipid matrix, thus allowing high EE and sustained release. Meanwhile, it was reported that the increased particle size of FPCN results in increasing the distance between the core and the surface . This isThe cellular uptake of different formulations was evaluated in MCF-7/ADR cells. The free paclitaxel showed poor cellular uptake of paclitaxel due to the p-gp efflux, while all of NPs prepared were easily taken up by the cells , 40. Co-3) is widely used as endocytosis inhibitor agent which is known to disturb the production of ATP in cells and block the endocytotic pathway [Next, flow cytometry analysis was conducted to figure out the cellular uptake mechanism for the FPCHN-30. Firstly, the effect of temperature on cellular uptake was studies by incubating the cells with FPCHN-30 at 37°C or 4°C for 4 h. As seen in Figure  pathway . In this pathway . In thisIn order to confirm the enhanced cellular uptake of paclitaxel and curcumin, cell cytotoxicity was measured by MTT assay. All the formulations exhibited higher cytotoxicity as compared to that of free paclitaxel and curcumin. In addition, folate conjugation onto surface of NPs allowed for further cytotoxicity. It was highlighted that FPCHN-30 exhibited still higher cytotoxicity compared to that of FPCN when the NPs were co-incubated with folate. FPCHN-30, rate-controlling and active-targeting delivery system, exhibited the highest cytotoxicity as well as cellular uptake on MCF-7/ADR cells.Finally, the p-gp inhibition efficiency of FPCHN-30 was evaluated by western blot assay and immunofluorescence staining. From the results obtained, FPCHN-30 exhibited maximum inhibition of p-gp among tested group. Similarly, to the results of cell studies, co-treatment of curcumin exhibited depressed p-gp expression as compared to that of paclitaxel alone. Specially, sequential release of curcumin and paclitaxel from FPCHN-30 was observed to have better inhibition of p-gp, strongly indicating the importance of release profile of both drugs and active targeting to achieve the enhanced therapeutic efficiency. The faster release of curcumin was able to inhibit p-gp efficiently so that paclitaxel could be easily internalized in MCF-7/ADR cells.On the basis of our experimental studies, FPCHN-30 could be used as a promising therapeutic delivery system for MDR-expressing breast cancer cells by sequential release of p-gp inhibitor and anticancer drug, and active targeting to FA receptors Figure .From this study, we presented a promising approach to improve the cellular uptake of paclitaxel and curcumin into MCF-7/ADR cells that express folate receptors and p-gp. Our novel system exhibited a significant enhanced cytotoxicity and cellular uptake from the combined effects of optimized sequential release of multiple drugs and folate receptor-mediated internalization by manipulating hydrophilicities and conjugation of folate with lipid. Since targeting ability and controlled release of curcumin and paclitaxel are achievable with FPCHN-30, this nanocarrier would have great potential to inhibit MDR-expressing tumor cells.Paclitaxel was provided by Samyang Genex . Stearic acid was purchased from Daejung Chemical . Poloxamer 188 was obtained from BASF  and lecithin was acquired from Junsei . Curcumin, HPCD, folate, DAPI, Nile Red and mannitol were purchased from Sigma-Aldrich . All other chemicals and reagents used were of analytical grade.Paclitaxel and curcumin loaded lipid nanoparticles (PCN) were prepared using emulsification and the solvent evaporation method . BrieflyFolate-conjugated curcumin and paclitaxel loaded SLNs (FPCN) were prepared by adding 5 mg of conjugated stearic acid and folate (SA-FA) in the organic lipid phase using the same method described above. The SA-FA was synthesized using EDC (1-ethyl-3(3-dimethylaminopropyl)carbodiimide) coupling reaction , 39. PreThe presence of folate in SA-FA on the particle surface was confirmed using X-ray photoelectron spectroscopy . The lyophilized nanoparticle samples were applied on a clean aluminum substrate and kept under vacuum. The surface chemical composition on the nanoparticles was examined using a surface spectrophotometer. XPS analysis was conducted for PCN and FPCN.18 column . The flow rate of the mobile phase was 1 mL/min and the detection wavelength of paclitaxel or curcumin was set to 227 nm or 425 nm, respectively. The mobile phase of paclitaxel or curcumin was a mixture of distilled water and acetonitrile  or distilled water and acetonitrile  adjusted pH 2.5 by formic acid, respectively. Drug loading and encapsulation efficiency (E.E) were calculated as follows:The lyophilized nanoparticles (10 mg) were solubilized with 1 mL of ethanol, heated at 80°C for 30 min and then cooled down at -20°C for 30 min. This solution was centrifuged at 10,000 g for 5 min to precipitate the undissolved solid lipid, filtered through a 0.22 μm filter and injected into the HPLC system. An Agilent 1100 liquid chromatography system with an autosampler and UV detector were used. The column used was a CDrug loading (%) = weight of the drug in particles/weight of the particles x 100.E.E (%) = weight of the drug in particles/weight of the feeding drugs x 100.The particle size and zeta potential analysis of different nanoparticles were performed by laser scattering analyzer . The lyophilized nanoparticles were dispersed in distilled water and sonicated to minimize the inter-particle interactions. The obscuration range was maintained between 20~50%. The instrument was set to measure the sample 100 times and the average volume mean diameter was obtained. The zeta potential of different nanoparticles was measured by Zetasizer Nano Z . Data were collected as the average of 20 measurements.In vitro release studies of different formulations were evaluated using a dialysis bag ), which was filled with an amount according to 20 mg of formulation. Then, dialysis bag was immersed in 20 mL of phosphate buffered solution (PBS) including 0.1% (w/v) of tween 80. Aliquots of 1 mL samples were withdrawn from the medium and replaced with the same volume of fresh dissolution medium at an indicated time. The concentrations of the released drugs were determined by HPLC as described above.2 at 37°C.MCF-7/ADR cell line was purchased from the American Type Culture Collection . The cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 100 units/mL penicillin in a humidified atmosphere of 5% CO6 cells per well. After 24 h incubation, the cells were further incubated with different formulations at different concentrations at 37°C. After the incubation period, the cell medium containing different formulations was removed from the wells and the cells were washed with cold PBS. The cells were lysed for 10 min by addition of 1% Triton X-100 (400 μL) per each well. After that, an aliquot of the cell lysate was used to measure the total cell protein amount by the BCA assay. The concentration of paclitaxel in the cell lysate was measured by HPLC as described above.To evaluate quantitative uptake of paclitaxel, the cells were seeded into a 6-well plate at the density of 1 × 106 cells per well in 1 mL of growth medium and incubated for 24 h to allow them to attach. The cells were then treated with the different formulations in growth medium. At predetermined time point (4 h), the cells were washed three times with cold PBS. Then, cells were stained with 1 μg/mL DAPI in PBS for 3 min and washed twice with PBS. The cells were observed directly under the CLSM.For cellular uptake of curcumin, the cells were incubated on glass base dish  and were examined by confocal laser scanning microscopy . In glass base dish, MCF-7/ADR cells were seeded at a density of 1 × 10In order to determine quantitative uptake of curcumin, the cells were washed three times with cold PBS, then collected by centrifugation and re-suspended in PBS (1 mL). The mean fluorescent intensity of curcumin in the cells was measured on a BD LSRFortessa flow cytometer .3) for 0.5 h. The effect of temperature on cellular uptake was assessed by incubation at 37 and 4°C.Flow cytometry analysis was adapted and four separate experiments were conducted including pretreatment of 450 mM of sucrose and 80 mM of sodium azide (NaN4 cells/mL (200 μL/well). After confluency, the cells were further incubated with the various formulations for 48 h. The culture medium was replaced by MTT solution , and the cells were incubated for a further 4 h. The supernatant was carefully removed, and 200 μL of DMSO was added to the each well. The plates were then placed in an incubator for 30 min. The absorbance values of each well were measured at 570 nm using a microplate reader .To determine cytotoxicity of the various formulations, the MTT assay (Sigma-Aldrich) was conducted. Briefly, MCF-7/ADR cells were seeded in 96-well plates at a cell density of 5×10TM HC . The membrane was blocked in 5% skim milk solution at room temperature for 1 h. P-gp (170 kDa) or β-actin (42 kDa) was detected by treating the samples with p-gp antibody C219  or β-actin antibody  at room temperature for 2 h, followed by IgG-HRP: sc-2004  as the secondary antibody at 4°C for overnight.MCF-7/ADR cells were lysed for 10 min and harvested by centrifugation at 15,000 g for 10 min. Aliquots from the suspension were resolved on a 10% sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) at 100 V for 120 min and electrotransferred onto a polyvinylidene difluoride (PVDF) membrane using a PowerPac6) cells were seeded on glass base dish  and incubated for 24 h to allow them to attach. The cells were then treated with the different formulations in growth medium. At predetermined time point (4 h), the cells were washed three times with PBS. Then, cells were treated with p-gp antibody C219  for 2 h, followed by Alexa 555 antibody 1:1000 dilutions, Thermo scientific, Rockford, USA). Then, cells were stained with 1 μg/mL DAPI in PBS for 3 min and washed twice with cold PBS. Fluorescence images of the cells were observed directly using the CLSM.In glass base dish, MCF-7/ADR (1 × 10t-test was used to compare the groups. A p < 0.05 as considered to indicate statistical significance. All data are expressed as the mean ± standard deviation from three independent experiments.Student’s


Acinetobacter lactucae OTEC-02 was isolated from hydrocarbon-contaminated soils. Whole-genome sequence analysis was performed to learn more about the strain’s ability to degrade different types of recalcitrant toxic monoaromatic hydrocarbons. The genome of this bacterium revealed its genomic properties and versatile metabolic features, as well as a complete prophage. Acinetobacter genus belongs to the gamma subclass of Proteobacteria. It comprises a diverse group of Gram-negative, strictly aerobic, nonfermenting, nonmotile organisms that are ubiquitous in nature and commonly found in soil .The  in soil . The strde novo genome assembly, which was completed using the program RS_HGAP_Assembly.3 with SMRT Portal Analysis version 2.3.0 (http://www.ncbi.nlm.nih.gov/genome/annotation_prok). Clusters of orthologous groups (COGs) were allocated using BLASTx against the COG database, and the hits were accepted with an E value of 1e10 (The OTEC-02 genome was sequenced using the Pacific Biosciences (PacBio RSII) single-molecule real-time (SMRT) sequencing platform. Four SMRT cells of a 15-to-20-kb insert library were sequenced; 168,961 reads, with an average read length of 10,793 bp, were used for the  of 1e10 . The OrtA. lactucae OTEC-02 has a single circular chromosome with an average G+C content of 38.8%. It contains 3,683 putative coding sequences , of which 784 have no COG prediction. The comparison between the OTEC-02 and A. lactucae NRRLB B-41902 strains showed that both genomes are very similar. The latter was isolated from lettuce in the United States , with which it shares only 10 proteins (A. lactucae strain OTEC-02 gives us the opportunity to study its capacity for being used in the bioremediation of contaminated soils.Soils contaminated with hydrocarbons represent an extreme environment. Strains that have the capacity to grow under these conditions have developed different strategies to survive. Analysis of the genome of A. lactucae OTEC-02 has been deposited in GenBank under the accession number CP020015.The whole-genome nucleotide sequence of


To study the concordance between vaginal self- and endocervical physician-collected high-risk (hr) HPV testing in Thai women who attended a colposcopy clinic. Vaginal samples were obtained by self-sampling with a dry brush before endocervical samples were obtained by physicians. Both specimens were analyzed for hrHPV by Cobas4800 HPV test.P < 0.001). Our study revealed moderate agreement between self- and physician-collected methods for hrHPV testing.Of the 247 pairs of samples, overall hrHPV prevalence from self- and physician-collected samples was 41.3 and 36.0%, respectively. The overall agreement between the methods was 74.5% with κ 0.46 ( Worldwide trends in incidence and mortality rate of cervical cancer have decreased as a result of effective organized screening programs, however, cervical cancer remains an important health problem in less-developed countries. In Thailand, it is the second most common cancer in women, with an age-standardized incidence rate of 17.8 per 100,000 .The Ministry of Public Health in Thailand launched a national screening program for women aged 30–60 years since 2002 but the coverage rate was 46–67%, which was lower than the target of 75% [Human papillomavirus (HPV) testing was approved for primary screening because of its satisfactory sensitivity for detecting high-grade precancerous cervical lesions –10. SelfDue to the uncommon use of tampon among Thai women, most of them are unfamiliar with inserting the device into their vagina. From our previous study, there was a concern that some women might not use the self-sample device properly . There hThe study protocol was approved by the Institutional Review Board of Chulabhorn Hospital (No. 10/2013). We recruited women aged 30–70 years who visited a colposcopy clinic at Chulabhorn Hospital, Bangkok, Thailand during March 1 to June 30, 2015. Women were eligible for inclusion if they were attending the colposcopy clinic, aged 30–70 years, had no history of cervical cancer, had not undergone a hysterectomy, and were currently not pregnant.After the participants had given written informed consent to participate in the study, they received instructions by video made by research project’s staffs to explain how to use the vaginal self-sampling brush, verbal and illustrations for vaginal self-sampling. The vaginal specimens were first obtained by self-sampling with the Evalyn Brush , which is a dry brush. Then, the participants were examined by a gynecological oncologist who obtained an endocervical sample with a Cervex-Brush .The self- and physician-collected specimens were both suspended in 10 ml transport medium, SurePath Preservative Fluid .All self-sampled and physician-collected specimens were sent to the central laboratory of Chulabhorn Hospital for hrHPV testing. All samples were analyzed by Cobas4800 HPV test  within 1 week after collection.Frequency, percentage, mean and standard deviation (SD) were used to calculate the general characteristics. The agreement levels were analyzed by Cohen’s kappa statistics. The statistical significance level was at 0.05. All data were analyzed by STATA/SE version 12.1.We enrolled 250 eligible women. Two participants were excluded because of a history of cervical cancer and previous hysterectomy. One pair of samples was excluded because of invalid test results. The mean and median ages of the remaining 247 participants were 47.2 and 47.0 years , respectively. Table Overall hrHPV prevalence was 41.3% 102/247) from self-collected specimens and 36.0% (89/247) from physician-collected specimens. The prevalence of hrHPV 16, 18 and non-16, 18 from self- and physician-collected specimens is shown in Table /247 fromThe concordance of hrHPV test results between self- and physician-collected specimens is shown in Table We found that the prevalence of hrHPV from self-collected specimens (41.3%) was higher than from physician-collected specimens (36.0%). To explain, the self-collected specimens are a combination of cervical and vaginal cells. Additionally, the sampling order first obtained from the self-collected specimens may then collect higher number of exfoliated cells. In particular, the higher prevalence of low-risk HPV in the lower vagina than in the cervix and scanty cross-reactivity of the hrHPV assay to low-risk genotypes can partially explain this finding , 29.Our study revealed 74.5% concordance between self- and physician-collected specimens in detecting overall hrHPV with κ 0.46, which showed moderate agreement. The level of agreement in our study was not as high as that in most previous studies –25. MostOne study showed that the agreement between these two methods was lower among older women, which supports our results . The medAlthough the agreement level of overall hrHPV between self- and physician-collected samples was moderate, the agreement level of HPV 16 was substantial, with concordance of 93.36% and κ 0.72. This finding for HPV 16 was the same as in the other studies , 25. As All the participants in our study did the self-collection first then underwent pelvic examination to obtain physician-collected specimens later. This sampling order may have resulted in the self-collected specimens having more exfoliated cells than the physician-collected specimens had.Our participants were women who attended a colposcopy clinic for various reasons such as abnormal cytology or positive HPV testing, so the prevalence of HPV in this group was higher than in the normal population. The prevalence of hrHPV in our study was 41.3 and 36.0% from self- and physician-collected specimens, respectively. The prevalence of hrHPV in Thai women in previous studies was 3.3–14.0% –36.Due to the level of agreement in our study was slightly lower than in most previous studies, more studies with larger populations are needed to explore the reliability and feasibility of self-sampling of HPV as a method for cervical cancer screening in Thai and other Asian women. The molecular and biomarker analyses may be combined to achieve greater accuracy of the test.


Circular RNAs (circRNAs) are a novel class of non-coding RNA which have recently shown huge capabilities in the regulation of gene expression at the post-transcriptional level. Growing evidence has indicated that circRNAs could serve as competing endogenous RNAs (ceRNAs) to bind with microRNAs (miRNAs) and to inhibit the activity and function of the targeted miRNAs. Here, we demonstrated that circ-0016347 acted as a positive regulator in osteosarcoma cells proliferation and invasion. Moreover, circ-0016347 was identified as a sponge of miR-214 that upregulated the expression of caspase-1, which is the functional target of miR-214. Our study provides novel insight into the regulatory mechanism of circ-0016347 and its downstream targets in proliferation, invasion and metastasis of osteosarcoma cells, which will facilitate further development in the therapy of osteosarcoma. Osteosarcoma is one of the most devastating malignant neoplasms that arises from primitive transformed cells of mesenchymal origin in children and adolescents . AlthougCircRNAs are a class of endogenous RNAs, which are characterized by covalently closed loop structures without polarity or a polyadenylated tail , 7. CircIn comparison to circRNAs, miRNAs have been extremely well studied. There is abundant evidence that miRNAs are involved in the occurrence and development of tumors, thereby being potential cancer biomarker candidates . A largeIt has been widely accepted that there is a strong association between inflammation and cancer. In the tumor microenvironment, inflammatory cytokines influence almost every aspect of tumor progression including invasion and metastasis abilities . CaspaseWe randomly collected osteosarcoma tissues and adjacent non-tumor tissues from 6 patients. The circ-0016347 expression levels were measured by qRT-PCR. The results showed that circ-0016347 expression levels were higher in osteosarcoma tissues than in the adjacent non-tumor tissues Figure . Moreovein vitro, we transfected pcDNA-circ-0016347 or pcDNA into the Saos-2 cells and then injected subcutaneously into the back of mice or the tail vein of mice. The results showed that the tumor sizes (E) or the numbers of pulmonary metastasis tumors (F) were distinctly increased in the circ-0016347 overexprssion mice than that in the control mices. Together these data indicated that circ-0016347 played a positive role in the proliferation, invasion and metastasis of osteosarcoma cells.To determine the functional role of circ-0016347 in the proliferation, invasion and metastasis of osteosarcoma cells, circ-0016347 siRNAs were used to knockdown circ-0016347 in MG-63 and Saos-2 cells, which have high endogenous circ-0016347 expression. Then, we assessed the effect of circ-0016347 on the proliferation and invasion of osteosarcoma cells. qRT-PCR revealed that circ-0016347 were significantly downregulated by circ-0016347 siRNA transfection compared with the negative siRNA control Figure . CCK-8 rA large body of evidence has documented that caspase-1 is important for the formation of the tumor microenvironment,contributing to the cell proliferation and invasion of osteosarcoma. Therefore, we attempted to analyze the relationship between circ-0016347 and caspase-1 in osteosarcoma. The previous results have shown that circ-0016347 levels were obviously elevated in six osteosarcoma tissues compared to matched adjacent non-tumor tissues Figure . ConsistMiRNAs, which regulate the stability and translational efficiency of partially complementary target mRNAs, are frequently aberrantly expressed in tumors, eventually promote proliferation in cell lines, and accelerate angiogenesis and tumorigenesis , 21. In Increasing evidence has have shown that circRNAs sequester miRNAs to terminate regulation of their target genes . Thus, wCirc-0016347 acts as a natural miR-214 sponge, and miR-214 has similar suppressive effects on the expression of circ-0016347. In Figure Osteosarcoma is the most common type of primary bone malignancy. Because of the lack of early diagnosis and effective therapeutic approaches, the mortality rates remain worryingly high for several years. Novel in-depth studies are essential to diagnose the osteosarcoma in earlier stages, which is the key to fighting against the disease.in vitro and in vivo. However, the specific function of circ-0016347 in the occurrence and development of osteosarcoma remains largely elusive.CircRNAs represent a novel class of regulatory non-coding RNA that have only recently been identified and cataloged. Emerging evidence has indicated that circRNAs are widely expressed in biological systems and are characterized by highly conserved sequences and a high degree of stability in mammalian cells . These pThere have been a number of studies on the complicated relationship between inflammation and cancer. It is well-known that chronic inflammation could promote the occurrence and development of tumors , 28. AddMicroRNAs play important roles in the regulation of various biological processes, including cell proliferation, apoptosis, metastasis and inflammation , 35. SpeEmerging researches show that there is functional crosstalk between circRNAs and miRNAs, but the effects of the interaction of circRNAs with miRNAs on the progression of osteosarcoma remain unknown. According to the prediction results of the bioinformatics software and previous results, we found that miR-214 could interact with both circ-0016347 and caspase-1. In addition, the expression level of miR-214 was obviously increased after circ-0016347 was inhibited. This suggests that circ-0016347 competitively binds to miR-214 and inhibitsmiR-214 activity, resulting in increased expression levels of the targeted gene of caspase-1. These results implied that circ-0016347 promoted osteosarcoma cell proliferation, invasion and metastasis, at least partly by influencing the formation of the inflammatory microenvironment through the miR-214/caspase-1 axis.In summary, our study demonstrates that the functional crosstalk between circ-0016347 and miR-214, as well as the down-stream target caspase-1, are critically involved in the proliferation, invasion and metastasis of osteosarcoma cells. Circ-0016347 acts as a miRNA sponge to directly inhibit the activity and function of miR-214 and then subsequently increases the expression of the down-stream target caspase-1 in osteosarcoma cells. This report revealed a novel mechanism of hsa-circ-0016347 and miR-214 in osteosarcoma. It might contribute to establishing potential therapeutic strategies for osteosarcoma.Six pairs of tissue samples were collected from patients diagnosed with osteosarcoma who underwent surgery at The First Affiliated Hospital of Harbin Medical University, China. The samples were snap-frozen and stored at −80°C until total RNA or protein extraction. All patients provided signed consent to the research. The Research Ethics Committee at Harbin Medical University approved the study.2 at 37°C. Cells were investigated within 8 h of harvest. Si-circRNAs against human circ-0016347 were constructed by RIBOBIO . Knockdown and overexpression of circ-0016347 and miR-214 were obtained from Invitrogen . All cell transfections were performed according to the manufacturer's protocol .Human osteosarcoma cell lines Saos-2 and MG-63 and the human osteoblast cell line hFOB (OB3) were purchased from the Chinese Cell Bank of the Chinese Academy of Sciences . Cells were cultured in Dulbecco's Modified Eagle Medium  supplemented with 10% (v/v) fetal bovine serum  in an atmosphere of 95% humidified air and 5% CO4 cells/well and maintained for 24 h. CCK-8 solution (10 μl) was added to each well and cells were incubated at 37°C for 2 h. The absorbance at 450 nm was evaluated using a microplate reader. The data are representative of three individual experiments carried out in triplicate.Cell proliferation was determined by the cell count kit-8 (CCK-8) cell proliferation kits according to the manufacturer's instructions. MG-63 and Saos-2 cells were seeded in 96-well plates at 1 × 10Osteosarcoma cells were seeded into six-well plates and grown to 80–90% confluence. A wound was produced by a straight scratch with a 200-μL sterile pipette tip. The osteosarcoma cells were then rinsed with phosphate-buffered saline (PBS) to remove the floating cells. Images were captured within 12h, 24 h post-wound. The relative distance of cell migration to the scratched area was measured and a healing percentage was calculated. Each test was carried out in triplicate for more than two independent experiments.5 cells/mL. After 24 h, Matrigel and cells remaining on the upper side of the membrane were wiped off, and the cells that had migrated to the bottom surface of the membrane were fixed in 4% paraformaldehyde in PBS. Once fixed, the cells were stained with crystal violet for 10 min at room temperature. Four randomly selected fields were captured using a fluorescence microscope  to calculate the number of cells that had successfully invaded and transmigrated the Matrigel. Data were expressed as the average number of cells per insert.Transwell chambers containing polycarbonate membrane filters with a 24-well 8-μm pore size  were coated with Matrigel. In each well, 40 μl of Matrigel was added to an insert and dried in a 37°C incubator for 30 minutes to form a thin gel layer. Then, 24 h after MG63 and Saos-2 cells transfected with si-circRNA, the cells were resuspended with reduced serum DMEM/F12 and were adjusted to 2.5 × 10The experimental were approved by the Ethic Committee of Harbin Medical University, China. Animal care and all animal related experimental procedures were conducted in accordance with the guidelines of the INHA Institutional Animal Care and Use Committee (INHA IACUC). Male BALB/c mice, 5 to 6 weeks old weighing 18-20 g were used. Firstly, Saos-2 cells were transfected with circ-0016347 overexpression plasmid (pcDNA-circ-0016347) or empty vector plasmid . After 48h, the Saos-2 cells were suspended in 100μl serum-free DMEM and then injected subcutaneously into the back of mice or injected into the tail vein of mice. After 2 weeks, the tumor were isolated and the longest diameters or the numbers of pulmonary metastasis tumors were calculated.−ΔΔCT method. For circ-0016347 and caspase-1,cDNA was synthesized using a reverse transcriptase kit . Real-time PCR was performed using the SYBR Green PCR Master Mix  and the ABI 7500 Sequence Detection System . PCR amplification was performed in a 20 μl reaction volume including 2 μl cDNA, 6 μl DEPC, 10μl SYBR Green Master Mix, 1 μl forward primer and 1 μl reverse primer. PCR conditions were as follows: 40 cycles of 95°C for 15 s, 60°C for 15 s, and 72°C for 45 s. Actin served as an internal control for circ-0016347 and caspase-1, and the results were calculated by using the 2−ΔΔCT method. The primers sequences are listed in Table Total RNA was extracted by Trizol reagent (Invitrogen). PCR primers were purchased from Invitrogen. The concentration of RNA was confirmed by a NanoDrop Spectrophotometer . For miR-214, cDNA was synthesized from 5 ng of total RNA by a TaqMan miRNA reverse transcription kit  to determine the miR-214 levels. The results were normalized to U6 levels and calculated by using the 2The luciferase reporter plasmid or empty vector was transfected into cells together with the encoding gene plasmid. Luciferase activities were measured at 48-h post-transfection using the Dual-Luciferase Reporter Assay System  according to the manufacturer's protocol. Renilla luciferase activity was normalized against firefly luciferase activity and presented as percent inhibition. All transfection experiments were performed in triplicate and repeated at least 3 times.Western blotting assays were used to detect the expression levels of caspase-1 proteins. The total protein of the tissues and cells was harvested with radioimmunoprecipitation assay buffer (RIPA) containing 1% protease inhibitor . For each sample, protein (100 μg) was separated using 12% SDS–PAGE and then transferred into nitrocellulose membranes . Afterwards, the membrane was blocked with 5% nonfat milk (BD Biosciences) and 0.1% Tween 20 in tris-buffered saline and immunoblotted overnight using caspase-1 primary antibodies at 4°C with gentle shaking. After that, a fluorochrome labelled secondary antibody  was used to identify the caspase-1 antibody. Immunoreactivity was detected with the Odyssey fluorescent scanning system (LI-COR) and analyzed by Image Studio software.To investigate the effect of circ-0016347 on the expression of caspase-1, cells were transfected with si-circ-0016347 or nonsense siRNA. After 24h, cells were fixed with 4% paraformaldehyde for 10 min at room temperature and washed with PBS before permeabilization by 0.5% Triton X-100 for 10 min. Nonspecific binding sites were blocked with 5% BSA containing 0.5% Trion X for 1 hour prior to incubation with caspase-1 antibodies overnight at 4°C. The cells were washed three times with PBS and incubated with secondary antibodies for 1 hour at room temperature. The nuclei were visualized by DAPI counterstaining for 5 min. Immunofluorescence images were observed utilizing an inverted fluorescencemicroscope .Data were expressed as the mean ± standard error of mean (mean ± SEM) and analyzed with SPSS 13.0 software. Statistical comparisons between two groups were performed using Student's t-test. Statistical comparisons among multiple groups were performed using analysis of variance (ANOVA). A two-tailed P < 0.05 was taken to indicate a statistically significant difference.


Stretching the patch along its width does not change the resonant frequency.In this paper, we propose a stretchable radio-frequency (RF) strain sensor fabricated with screen printing technology. The RF sensor is designed using a half-wavelength patch that resonates at 3.7 GHz. The resonant frequency is determined by the length of the patch, and it therefore changes when the patch is stretched. Polydimethylsiloxane (PDMS) is used to create the substrate, because of its stretchable and screen-printable surface. In addition, Dupont PE872  silver conductive ink is used to create the stretchable conductive patterns. The sensor performance is demonstrated both with full-wave simulations and with measurements carried out on a fabricated sample. When the length of the patch sensor is increased by a 7.8% stretch, the resonant frequency decreases from 3.7 GHz to 3.43 GHz, evidencing a sensitivity of 3.43 × 10 Strain sensors detect deformation and perform measurements when force is applied on them. Semiconductor-based sensors have a higher sensitivity and are lower in price than nanomaterial-based sensors ,2. NeverDue to high demands on wearable and bio technologies, the importance of nanomaterial-based strain sensors is increasing. In addition, soft and flexible materials such as polydimethylsiloxane (PDMS) and Eco-flex have been widely studied in strain sensors based on nanomaterials  ,6,7,8. FIn this study, we use a screen printing method on an elastomer for radio-frequency (RF) strain sensor applications. Screen printing technology is a good candidate for sensor applications because of its simple fabrication and low-cost mass production ,16. In oThe proposed strain sensor was designed based on a rectangular patch resonator. Rectangular patches are widely used for RF resonators and resonator-based components, because of their simple design and easy fabrication. In this work, the conductive patterns were created using screen printing technology, and the surface resistance of the conductive pattern was determined based on the width-to-length ratio. The surface resistance of the stretchable silver ink is 0.65 Ω and rectangular patch is 14 Ω. The lower surface resistance of the rectangular patch resonator is achieved due to lower width-to-length ratio. rε) and loss tangent were determined using a T-resonator method [εeff is the effective dielectric constant resulting from the fringe fields and Hs is the thickness of the substrate. The values of rε and loss tangent of the PDMS were determined to be 3 and 0.02, respectively. In order to characterize dielectric constants and loss tangent of the PDMS, we used T-resonator technique, as introduced in [pW) and length (pL) of the rectangular patch were therefore set at 17 mm and 23 mm, respectively, to allow the device to resonate at 3.7 GHz. pL of the patch, we expect the resonant frequency to decrease when the patch is stretched. pW and pL, respectively. Initially, the unstretched patch sensor resonates at 3.7 GHz, with a −25 dB reflection coefficient. As shown in pW is varied [As mentioned, PDMS—a stretchable dielectric material—was used as substrate for the patch resonator. Its dielectric constant fduced in ,20. The sW), length (sL), and thickness (sH) of the PDMS were 40, 50, and 1 mm, respectively. Having completed this, a liquid composition of a mixing base and a curing agent at a 10:1 ratio was prepared; a vacuum deaerator was used to remove the bubbles formed during the mixing process. The thick liquid composition can be cured at 25 °C for 48 h, or at 100 °C for 45 min. We performed heat curing at 80 °C for 30 min, using a hot plate. Next, for plasma treatment, we used the PDC-32G plasma cleaner from Harrick Plasma in U.S.A. Energy of plasma treatment for 20 s is 18 W.The fabricated RF strain sensor is shown in x- and y-directions, as shown in the inset of x-direction. As expected from the simulation results in y-direction. As expected from the simulation results in In addition, the reflection coefficient was measured at different strains. The fabricated patch prototype was stretched along the y-direction (length) is plotted in x-direction, as shown in y-direction, as shown in y = −0.0343x + 3.7. Therefore, the sensitivity of the proposed RF strain sensor is 3.43 × 107 Hz/%. The performance of the proposed strain sensor is compared with other strain sensors in The relation between the resonant frequency and the strain along the x-direction (width) and 7 Hz/% was therefore obtained. In contrast, the resonant frequency did not change when stretches were applied along the width dimension of the patch sensor. It can therefore be concluded that the proposed screen-printed RF patch resonator enables low-cost, high-volume fabrication of strain sensors.In this study, a screen-printed RF strain sensor using stretchable silver ink and PDMS was proposed. A rectangular patch resonator was designed and used as an RF strain sensor by considering the change in resonant frequency that results from stretches applied along the patch length dimension. The feasibility of this device was demonstrated using both simulation and measurements. Initially, the unstretched patch sensor resonated at 3.7 GHz with a −28 dB reflection coefficient. When the length of the patch sensor was increased by a 7.82% strain, the resonant frequency decreased from 3.7 GHz to 3.43 GHz. A sensitivity of 3.43 × 10


Atopic dermatitis (AD) is a common chronic inflammatory skin disorder, and patients with AD suffer from severe psychological stress, which markedly increases the prevalence rate of depression and anxiety disorders in later life. Fluoxetine, a selective serotonin reuptake inhibitor, has recently been reported to exert anti-inflammatory and immunosuppressive effects. However, it is unclear whether fluoxetine is effective in the treatment of AD through reducing psychological stress and inflammatory reaction. Here, we reported that a BALB/c mouse model of AD was induced by application of 2,4-dinitrochlorobenzene (DNCB) onto hairless dorsal skin. Chronic fluoxetine treatment  significantly attenuated AD-like symptoms, as reflected by a dramatic decrease in scratching bouts, as well as a decrease in anxiety- and depressive-like behaviors. Furthermore, these behavioral changes were accompanied by a significant decrease in epidermal thickness, the number of mast cells in skin tissue, mRNA levels of interleukin-4 (IL-4) and IL-13 in the spleen, as well as serum immunoglobulin E (IgE) in the DNCB-treated mice by treatment with fluoxetine. Taken together, these results indicate that fluoxetine may suppress psychological stress and inflammatory response during AD development, and subsequently ameliorate AD symptoms, suggesting that fluoxetine may be a potential therapeutic agent against AD in clinic. Atopic dermatitis (AD), also known as atopic eczema, is a common chronic inflammatory skin disease that is characterized by intense itching and recurrent eczematous lesions. The prevalence rate of AD is rising dramatically, especially in developed countries, and it now affects 10–30% of children and 1–10% of adults , leadingin vitro and in vivo  colony room on a 12/12 h light/dark cycle. Food and water were available Fluoxetine and DNCB were purchased from Sigma-Aldrich Co. . DNCB was dissolved in acetone-olive oil  and fluoxetine was dissolved in 0.9% sterile saline at a concentration of 10 mg/ml. The drug application and experimental tests were performed in a double-blinded manner.Figure 1 as described previously .Induction of AD-like skin lesions procedure is described in eviously . The bacThe severity of DNCB-induced skin lesions was clinically assessed as previously described . BrieflyThe animals were placed into an observation chamber for 10 min for acclimatization before the measurement of scratching behavior. The scratching behavior was defined as the hind limb scratches directed to the shaved area. The number of scratching behaviors was counted for 30 min each time.Mice were placed in the center of a plus maze (each arm 30 cm) that was elevated 50 cm above the floor with two opposite open arms and two opposite closed arms  arranged at right angles. The number of entries and time spent in the closed and open arms were monitored for 10 min by ANY-Maze Video Tracking System. The maze was cleaned with 70% ethanol and water between tests.Mice were placed in a cylinder of water  for 10 min. The depth of water was set to prevent animals from touching the bottom with their hind limbs. The latency to immobility, which was defined as floating or the least movement to maintain the head above the water, was recorded by ANY-Maze Video Tracking System (Stoelting Co.) from the side.A portion of the skin biopsies were fixed in 4% paraformaldehyde. The skin sections (4 μm thick) were stained with hematoxylin and eosin (H&E) to evaluate the epidermal hyperplasia and the other sections were stained with toluidine blue to evaluate the infiltration of mast cells. Mast cells were counted in five parts of high-power fields (HPF) at 400× magnification.Blood samples were collected by cardiac puncture under anesthesia, and sera were collected by centrifugation and stored at -80°C until use. Total IgE levels in plasma were determined by sandwich ELISA using the Sigma mouse IgE ELISA kit .® Premix Ex TaqTM II . Primer sequences were as follows: Interferon-γ (IFN-γ) ; IL-2 ; IL-4 ; IL-13 ; β-actin . The relative expression levels of target genes were normalized using β-actin as an internal control.Total RNA was extracted from spleen tissue with the TRIzol reagent  according to the manufac turer’s instruction. The quantity and purity of total RNA were determined with a Nanodrop reader . One microgram of total RNA was converted to the first-strand DNA with PrimeScript RT reagent Kit with gDNA Eraser  reverse transcriptase  and RNAsin . cDNA was amplified using gene-specific primers and SYBRp < 0.05.All data are expressed as the mean ± SEM. Statistical analysis of the results were performed by one-way analysis of variance (ANOVA) followed by Turkey’s test. Significance level was set at Figure 1). Consistent with previous reports . More importantly, chronic fluoxetine administration dramatically alleviated skin lesions . To further compare the skin lesions of the mice with those of human AD . In addition, chronic fluoxetine treatment significantly suppressed DNCB-induced scratching behaviors . Taken together, these results suggest that chronic fluoxetine treatment alleviates the DNCB-induced skin lesions.Previous studies have shown that paroxetine, a selective SSRI, is effective in the treatment of AD in both animal model and patients, with no apparent side-effects . To dete reports , AD-likehuman AD , clinicahuman AD . The resn = 10, 186.8 ± 5.0 s; DNCB: n = 10, 157.2 ± 10.4 s, p < 0.05 vs. control; Figure 3A). In agreement with an anti-depressive effect of fluoxetine, it succeeded in abolishing the influence of DNCB on depressive behavior .A growing body of evidence has shown that AD in both adolescence and adulthood increases the risk of developing major depression and anxiety disorders in later life , and flun = 10, 25.5 ± 3.9; DNCB: n = 10, 11.1 ± 1.5, p < 0.05 vs. control; Figure 3B) and the time spent in open arms  were significantly reduced in DNCB-treated mice during elevated plus maze test. Similar to forced swimming test, chronic fluoxetine treatment was able to restore the number of entry into the open arms  and the time in open arms  to control levels, indicating chronic fluoxetine administration reduces anxiety during AD development.Next, using elevated plus maze paradigm, we tested the effect of fluoxetine on anxiety-like behavior in AD. The results showed that both the number of entry into open arms . Similarly, the number of mast cells in the dermis of DNCB-treated mice was markedly increased as compared to the normal group, whereas this increment was significantly suppressed by chronic fluoxetine treatment .It has been widely accepted that AD is a common chronic inflammatory skin disease. We next wanted to determine the alterations of tissue inflammation and the accumulation of mast cells in AD-like skin lesions. Consistent with previous reports , histopaFigure 5, the serum level of IgE was markedly increased in the DNCB-treated group compared to normal control . Chronic fluoxetine treatment restored the serum IgE to normal control level .Since serum IgE level is correlated with the severity of AD , and then = 4, 100.0 ± 14.7; DNCB: n = 4, 288.6 ± 7.1, p < 0.01 vs. control; Figure 6A) and IL-13  mRNA expressions were dramatically increased, whereas the IL-2  and IFN-γ  mRNA expressions remained unchanged in DNCB-treated mice as compared to the normal group. More importantly, chronic fluoxetine treatment suppressed the DNCB-induced increase of IL-4  and IL-13 mRNA expressions In the past decades, SSRIs including fluoxetine have been shown to exert several immunological effects, such as alteration of cytokine secretion , 2014. TIn the present study, we confirm that repeated DNCB treatment induces the AD-like pathology, and demonstrate that chronic fluoxetine treatment reduces DNCB-induced inflammatory response and subsequently ameliorate AD symptoms in BALB/c mice. Therefore, our current results indicate that fluoxetine may effectively prevent AD symptoms.2+ signaling in Jurkat T lymphocytes through depletion of Ca2+ from intracellular stores . Notably, chronic treatment with fluoxetine impairs adult neurogenesis and locomotor activity in mice (Alternatively, fluoxetine may modulate psycho-emotion and subsequently prevent AD symptoms. Previous study has shown that the majority of patients with AD are subjected to psychological stress, and in turn, stress aggravates their pruritus, suggesting that pruritus might be closely related with emotional distress in AD . Fluoxet in mice . Thus, fIn summary, we demonstrate that chronic fluoxetine treatment is an effective means to reduce DNCB-induced scratching bouts, depressive/anxiety-like behaviors, skin lesions and serum IgE levels in mice model of AD, and these effects are mediated by regulating pro-inflammatory cytokines, such as IL-4 and IL-13. These results suggest that fluoxetine may be a potential therapeutic agent against AD in clinic; nevertheless, further investigations would be necessary to clarify its molecular mechanisms of action in AD.YL, LC, YD, and DH performed the research. HH and ZD designed the research study. YL, HH, and ZD contributed essential reagents or tools. YL, LC, YD, and ZD analyzed the data. HH and ZD wrote the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.